Project Summary This proposal seeks to build on our extensive preliminary data in canine immunotherapy trials to demonstrate that novel methods of exogenous cytokine delivery in combination with autologous natural killer (NK) cells in first-in-dog clinical trials represents a potentially high impact approach to optimize non-T cell based immunotherapies. This work is targeted to dogs with typically lethal, naturally occurring osteosarcoma (OSA) and melanoma, and the findings are expected to have important relevance for the design and translation of innovative immunotherapy approaches in humans. Inhaled (IH) IL-15 offers the advantages of local delivery of this immune-stimulatory cytokine, while limiting systemic exposure and thus potential toxicity. Super-agonist IL- 15 offers increased half-life and greater anti-tumor effects. Therefore, each of these cytokines is anticipated to alter the risk/benefit ratio in favor of their use. To accomplish these objectives, we propose the following three specific aims: 1) Targeting gross pulmonary metastases (OSA and melanoma) with first-in-dog delivery of inhaled (IH) human IL-15 and super-agonist IL-15; 2) Phase II trial of IH IL-15 and autologous NK cells to treat gross OSA and melanoma pulmonary metastases, and; 3) Targeting micro-metastatic disease using super- agonist IL-15 in primary OSA. The canine model represents a powerful tool in cancer immunotherapy research as an important link between murine models and human clinical studies. Dogs represent an attractive outbred combination of companion animals that experience spontaneous cancer development in the setting of an intact immune system. Importantly, these studies are designed to evaluate novel treatment combinations for advanced pulmonary metastases in the setting of gross disease, but also will determine the impact of this approach as first-line therapy when combined with standard-of-care treatments. Moreover, this work seeks to elucidate the cellular and molecular mechanisms that mediate an anti-tumor response (or non-response) in dogs with naturally occurring cancers. We will have the unique opportunity to perform whole exome sequencing and RNASeq on tumor tissue pre- and post-immunotherapy, and we will investigate whether a signature of mutational landscape and gene expression profiles can be developed to prospectively predict outcome and response to immune therapies. We will correlate these data to immunohistochemical, flow cytometry, and serum cytokine readouts for evidence of transition from ?cold? to ?hot? tumors predictive of favorable long term outcome. Hence, these canine trials and bio-marker studies represent an ideal strategy to inform and facilitate the rapid translation of novel, potentially high impact immune therapies to human patients with aggressive cancers.